Transplantation of neuron‐inducing grafts embedding positively charged gold nanoparticles for the treatment of spinal cord injury

Abstract In this study, we aimed to investigate the recovery after traumatic spinal cord injury (SCI) by inducing cellular differentiation of transplanted neural stem cells (NSCs) into neurons. We dissociated NSCs from the spinal cords of Fisher 344 rat embryos. An injectable gel crosslinked with glycol chitosan and oxidized hyaluronate was used as a vehicle for NSC transplantation. The gel graft containing the NSC and positively charged gold nanoparticles (pGNP) was implanted into spinal cord lesions in Sprague–Dawley rats (NSC‐pGNP gel group). Cellular differentiation of grafted NSCs into neurons (stained with β‐tubulin III [also called Tuj1]) was significantly increased in the NSC‐pGNP gel group (***p < 0.001) compared to those of two control groups (NSC and NSC gel groups) in the SCI conditions. The NSC‐pGNP gel group showed the lowest differentiation into astrocytes (stained with glial fibrillary acidic protein). Regeneration of damaged axons (stained with biotinylated dextran amines) within the lesion was two‐fold higher in the NSC‐pGNP gel group than that in the NSC gel group. The highest locomotor scores were also found in the NSC‐pGNP gel group. These outcomes suggest that neuron‐inducing pGNP gel graft embedding embryonic spinal cord‐derived NSCs can be a useful type of stem cell therapy after SCI.


Preparation of nGNP
HAuCl4 powder (136 mg) was dissolved in 800 ml of DW. Three hundred mg of citrate powder were mixed with 15 ml of DW to make citrate solution. The HAuCl4 solution was refluxed at 110 °C and the citrate solution (15 ml) was quickly added to the HAuCl4 solution. After 20 min, the color of the solution became dark red, containing approximately 30 nm of negative GNPs. The concentration of synthesized nGNP was 0.52 nM, which was estimated by the Beer-Lambert law with a molar extinction coefficient of 3.36 x 10 9 M -1 cm -1 .

Preparation of 2% gC and 3% oHA
The oHA was synthesized through an oxidation process using sodium periodate (NaIO4). The HA (3.8 g) was dissolved in DW (360 ml). The sodium periodate (1.068 g) was dissolved in another amount of DW (40 ml). The dissolved sodium periodate was slowly added to the HA in a dark room. The mixture was stirred for 24 h and 1 mL of ethylene glycol (Sigma) was added to the mixture to neutralize the unreacted sodium periodate. The resultant solution was dialyzed for seven days using dialysis membranes (Spectrum Spectra, molecular weight cut off (MWCO): 12-14 K). After dialysis, the oHA solution was lyophilized. The 2% gC and 3% oHA were separately dissolved in DPBS and centrifuged to remove the bubbles for 30 seconds at 13,000 RPM. These samples were stored at -20 °C before the use.

qRT-PCR
NSCs (4 × 10 6 ) embedded with 20 µl of hydrogels (n = 3 per group) were cultured with none (for gel group), 0.1 nM nGNP (for nGNP gel group), or 0.1 nM pGNP (for pGNP gel group). The NSC gels were cultured with medium for six days. The RNA in the groups was extracted using Trizol reagent (Invitrogen) according to the manufacturer's instructions. Complementary DNA (cDNA) was synthesized from 1 μg of total RNA using a Maxim RT Premix kit (iNtRON Biotechnology, Inc., Korea).
The qRT-PCR step was performed with an ABI Step-One real-time PCR system (Applied Biosystems, Warrington, UK). The reaction mixture consisted of the SYBR Green 2X PCR Master Mix (Applied Biosystems), a cDNA template, and forward/reverse primers. The relative expression levels of Tuj1, GFAP, and Olig2 were normalized to that of GAPDH using the 2 -ΔΔCT method. The primers were obtained from Bioneer (Daejeon, Korea). The primer sequences used in this study are shown in Table   S1. The experiments were conducted in triplicate.

Tissue preparation and IF staining for the measurement of the injury area
Two sections (5 μm thick) per specimen (n = 8 per group) were stained with anti-CD68/GFAP. The injury area surrounded an astrocytic barrier (stained with GFAP, mouse, Abcam at 1:200) is indicated by the white dashed lines. The ROI areas (composite tiled scan images, 4,500 × 2,000 μm 2 , scale bar: 500 µm) were normalized to 100% and relative injury areas were quantified. For the quantification of CD68 (rabbit, Abcam at 1:200 at 1:200) stained area, the injury area was normalized to 100% and the CD68-stained areas within the injury areas were quantified using ImageJ software (NIH). Alexa 488/568-secondary antibodies (1:500, Invitrogen) were used and the sections were detected through a confocal laser-scanning microscope (LSM 880, Carl Zeiss).